Whether pathogenic or opportunistic, microorganisms have evolved numerous mechanisms to facilitate their establishment and proliferation in mammalian hosts. During initial infection, the interaction of a microorganism with its mammalian host can include attachment or adhesion to the host cell surface, and invasion of host cells, for example. In certain instances, this interaction can be nonspecific. In others, such microbial interaction involves the specific binding of the microorganism to a particular receptor or receptor complex expressed on the host cell surface. In turn, the binding event can trigger changes in the microorganism and/or the mammalian host cell, leading to the progression of infection.
Candida is an ubiquitous yeast recognized as the causative agent of candidiasis (Candida mycosis). At least 90% of the disorders are caused by the species in normal individuals. However, destabilization of the host-parasite equilibrium upon inopportune loss or deficiencies in protective innate and immune deterrents favors overgrowth of the common gastrointestinal tract denizen and opportunistic pathogen, C. albicans. Acquired immunodeficiency syndrome (AIDS) or iatragenic immunosuppression are risk factors for oropharyngeal and esophageal candidiasis (Hood et al., 28 CLIN. INFECT. DIS. 587-96 (1999)). Thus, oropharyngeal and esophageal candidiasis are among the most frequent opportunistic fungal infections observed in human immunodeficiency virus positive (HIV+) and AIDS patients, occurring in the majority of patients. Candidal infections increase in severity and recur more frequently as the immunodeficiency progresses. The current status of the AIDS epidemic is one of increasing numbers of individuals infected and no cure. Many infected individuals may live for a long time with HIV in an essentially permanent immunocompromised state. Because of the loss of the cellular component of the immune system, AIDS patients are susceptible to invasion of submucosal tissue by C. albicans. In addition to HIV infected patients, oral candidiasis occurs in patients with leukemia or other cancers, as well as in patients with other underlying diseases. Prematurely-born infants are also at risk and may acquire mucosal infections causing permanent sequelae (Huang et al., 30 SCAND. J. INFECT. DIS. 137-42 (1998); Sood et al., 41 MYCOSES 417-9 (1998)). Candidiasis in denture wearers, or denture stomatitis, is the most common of all C. albicans associated diseases.
Although C. albicans is sensitive to antifungal drugs, treatment over long periods of time is required. At present, the treatment for invasive infections is based on relatively few antimycotics. Nystatin, ketoconazole, and amphotericin B are drugs, which are used to treat oral and systemic Candida infections. However, orally administered nystatin is limited to treatment within the gut and is not applicable to systemic treatment. Some systemic infections are susceptible to treatment with ketoconazole or amphotericin B, but these drugs may not be effective in such treatment unless combined with additional drugs. Amphotericin B has a relatively narrow therapeutic index and numerous undesirable side effects and toxicities occur even at therapeutic concentrations. While ketoconazole and other azole antifungals exhibit significantly lower toxicity, their mechanism of action, inactivation of cytochrome P450 prosthetic group in certain enzymes (some of which are found in humans), precludes use in patients that are simultaneously receiving other drugs that are metabolized by the body's cytochrome P450 enzymes. See, e.g., U.S. Pat. No. 5,863,762.
Other known antifungal agents include: polyene derivatives, such as amphotericin B (including lipid or liposomal formulations thereof) and the structurally related compounds nystatin and pimaricin; flucytosine (5-fluorocytosine); azole derivatives (including ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, tioconazole, terconazole, fluconazole, itraconazole, voriconazole [Pfizer, Groton, Conn.] and SCH56592 [Schering-Plough, Kenilworth, N.J.]); allylamines-thiocarbamates (including tolnaftate, naftifine and terbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins (including MK-0991 [Merck, Whitehouse Station, N.J.]); nikkomycins; and bactericidal/permeability-increasing protein (BPI), as described in U.S. Pat. Nos. 5,627,153; 5,858,974; 5,652,332; 5,763,567; and 5,733,872. Unfortunately, antimycotics cause serious, sometimes different, side effects, such as renal insufficiency, hypocalcemia and anemia, as well as unpleasant constitutional symptoms such as fever, shivering and low blood pressure.
The frequency of candidal infections may be a result of the prophylactic use of antibacterial drugs used in AIDS patients to minimize other opportunistic infections. Emergence of drug-resistant isolates and the limited selection of antifungal drugs point to the need for research aimed at identifying new anti-fungal targets (Terrell, 74 MAYO CLIN. PROC. 78-100 (1999)). However, the pathogenesis is complex and is thought to involve multiple host factors that include loss of cell mediated immunity and altered phagocytic cell activity. High frequencies of nosocomial candidemia reflect the ability of C. albicans to translocate across the gastrointestinal tract, disrupting internal tissues in debilitated patients (Viscoli et al., 28 CLIN. INFECT. DIS. 1071-9 (1999)).
Thus far, studies have shown that development of candidiasis is a multi-stage process requiring sensing environmental conditions and transducing signals to regulate expression of appropriate genes at balanced levels in C. albicans. Filamentous growth of C. albicans includes not only pseudohyphal, elongated yeast-like forms described for Saccharomyces cerevisiae, but true hyphae as well. Compared to most pathogenic fungi, the morphological response of C. albicans to environmental conditions is rapid. Germ tubes are produced within one hour of placing cells in appropriate conditions. The mechanisms employed by C. albicans to achieve this apparently advantageous spectrum of growth morphologies and optimized metabolic activities are poorly understood.
A feature of C. albicans growth that is correlated with pathogenicity in the oral cavity is the ability to transform from budding to filament-extending growth. Filamentous forms adhere more readily to buccal epithelial cells than budding yeasts, and histologically are a prominent feature of invasion of the mucosa. In mucosal disease, filamentous forms, particularly true hyphae, invade the keratinized layer of differentiated, stratified squamous epithelium. True hyphae are septate, cylindrical structures with parallel sides that are formed by extension of germ tubes that emerge from yeasts in appropriate environmental conditions.
The relative contribution of yeast and filamentous forms to the pathogenesis of candidiasis is an unresolved issue. However, mutants that do not produce hyphae in vitro have reduced virulence in animal models (Ghannoum et al., 63 INFECT. IMMUN. 4528-30 (1995); Lo et al., 90 CELL 939-49 (1997); Sobel et al., 44 INFECT. IMMUN. 576-80 (1984)). Expression of hypha-specific virulence factors such as the hyphal wall protein (HWP1) adhesin gene (Staab et al., 283 SCIENCE 1535-38 (1999); Staab et al., 271 J. BIOL. CHEM. 6298-305 (1996)) and secreted aspartyl proteinase (SAP) genes (Schaller et al., 34 MOL. MICROBIOL. 169-80 (1999); Staab et al., 97 PROC. NATL. ACAD. SCI. USA 6102-7 (2000)) are correlated with the virulence of hyphal forms. Research into the mechanisms that lead to the production of these virulence factors is important for developing strategies to interfere with candidiasis.
Thus, an alternative method to the prevention and treatment of candidiasis may be approached via disruption of molecular events that transform C. albicans to the pathogenic filamentous form. In many pathogenic fungi, interconversions between morphological growth forms, particularly between yeast growth and filamentous growth, coincide with adaptation to a host environment followed by tissue destruction. Morphological transitions are accompanied by expression of virulence attributes for many pathogenic fungi. A central pathway that regulates these transitions is the conserved cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, which modulates yeast and pseudohyphal growth of the model non-pathogenic yeast, Saccharomyces cerevisiae (D'Souza and Heitman, 25 FEMS MICROBIOL. REV. 349-364 (2001); Kronstad et al., 170 ARCH. MICROBIOL. 395-404 (1998); and Lengeler et al., 64 MICROBIOL. MOL. BIOL. REV. 746-785 (2000)). In response to specific environmental cues, S. cerevisiae transmits signals to the adenylate cyclase complex through a GTP-binding protein (G protein, Gpa2 and Gpb1/Gpb2) associated with a G protein-coupled receptor (Gpr1) or small G protein such as Ras (Gimeno et al., 68 CELL 1077-1090 (1992); Harashima and Heitman, 10 MOL. CELL BIOL. 163-173 (2002); Kübler et al., 272 J. BIOL. CHEM. 20321-20323 (1997); Lorenz et al., 154 GENETICS 609-622 (2000)). The adenylate cyclase complex of S. cerevisiae produces cAMP and is composed of adenylate cyclase (Cyr1) (Matsumoto et al., 79 PROC. NAT. ACAD. SCI. 2355-2359 (1982) and the adenylate cyclase-associated protein (CAP/Srv2)(Fedor-Chaiken et al., 61 CELL 329-340 (1990); and Gerst et al., 11 MOL. CELL BIOL. 1248-1257 (1991)). cAMP binds to the regulatory subunit (Bcy1/Sra1) of PKA (Cannon and Tatchell, 7 MOL. CELL BIOL. 2653-2663 (1987); Kunisawa et al., 15 NUCL. ACIDS RES. 368-369 (1987)), releasing the active catalytic subunits Tpk1, Tpk2, and Tpk3 from PKA. Although these catalytic subunits are functionally redundant in many cellular processes, Tpk2 is a positive regulator of filamentous growth, whereas Tpk1 and Tpk3 play an inhibitory role (Pan and Heitman, 19 MOL. CELL BIOL. 4874-4887 (1999)). Negative regulation of the cAMP-signaling pathway by S. cerevisiae occurs when two cAMP phosphodiesterases, Pde1 (low-affinity) and Pde2 (high-affinity), hydrolyze cAMP to AMP and restore PKA to the inactive state (Nikawa et al., 7 MOL. CELL BIOL. 3629-3636 (1987); and Sass et al., 83 PROC. NAT. ACAD. SCI. 9303-9307 (1986)). Pde2 is implicated in filamentous growth in that exogenous cAMP enhances production of pseudohyphae in pde2 mutants (Lorenz and Heitman, supra). Downstream targets of the cAMP/PKA pathway include a transcription factor, Flo8, and a cell wall flocculin, Flo11p, that modulates pseudohyphal differentiation, invasive growth, and cell-cell adhesion of S. cerevisiae (Gagiano et al., 31 MOL. MICROBIOL. 103-116 (1999); Guo et al., 97 PROC. NAT. ACAD. SCI. 12158-12163 (2000); and Rupp et al., 18 EMBO J. 1257-1269 (1999)).
Thus, an emerging theme of pathogenesis for plant and animal pathogenic fungi is that modulation of the cAMP dependent signaling pathway is required for morphological transitions to forms expressing virulence attributes necessary for attachment and invasion of host tissues. Induction of virulence gene expression may be accompanied by a morphological transition; however, the morphological form carrying the virulence attributes varies among fungi. In Magnaporthe grisea, the CPKA and MAC1 genes, encoding the PKA catalytic subunit and adenylate cyclase respectively, are required for appressorium formation (Choi and Dean, 9 PLANT CELL. 1973-1983 (1997); Mitchell and Dean, 7 PLANT CELL. 1869-1878 (1995)) and subsequent invasion of the hydrophobic surface of the plant. A mutant lacking the MAGB gene encoding the α-subunit of G protein was found to be defective in conidiation and appressorium formation (Liu and Dean, 10 MOL. PLANT MICROBE. INTERACT. 1075-1086 (1997)). In the corn smut fungus, Ustilago maydis, null mutants in any component of the cAMP-signaling pathway cause defects in morphological conversions and in pathogenesis (Gold et al., 8 GENES DEV. 2805-2816 (1994); Kruger et al., 13 MOL. PLANT MICROBE. INTERACT. 1034-1040 (2000); and Regenfelder et al., 16 EMBO J. 1934-1942 (1997)). In the case of Cryptococcus neoformans, activation of the cAMP dependent signaling pathway by the G protein α-subunit, Gpa1, and the PKA catalytic subunit, Pka1, leads to production of the virulence factors, such as capsule and melanin, on yeasts without changing cell morphology (Alspaugh et al., 11 GENES DEV. 3206-3217 (1997); and D'Souza et al., 21 MOL. CELL BIOL. 3179-3191 (2001)). Whereas genes that serve to activate the cAMP pathway are required for virulence in pathogenic fungi in general, the effects of genes that down-regulate the pathway are poorly understood.
The theme of activation of the cAMP dependent signaling pathway to express virulence attributes necessary for attachment and invasion of host tissue extends to the human fungal pathogen Candida albicans. A pulse of cAMP that requires the CAP1 gene (Bahn and Sundstrom, 183 J. BACTERIOL. 3211-3223 (2001)) in response to germ tube inducing conditions leads to production of germ tubes and surface expression of the germ tube specific adhesin, Hwp1, which is required for mucosal and systemic candidiasis (Staab et al., 283 SCIENCE 1535-1538 (1999); Sundstrom et al., 185 J. INFECT. DIS. 521-530 (2002a); and Sundstrom et al., 70 INFECT. IMMUN. 3281-3283 (2002b)). Other studies have found that the CaCDC35 and Tpk1/2 genes, encoding adenylate cyclase and two catalytic subunits of PKA respectively, are required for filamentous growth and virulence of C. albicans (Bockmühl et al., 42 MOL. MICROBIOL. 1243-1257 (2001); and Rocha et al., 12 MOL. BIOL. CELL 3631-3643 (2001)). For C. albicans, activation of the cAMP-signaling pathway promotes invasiveness and adherence as a consequence of the growth as germ tubes and hyphae.
Although studies of the cap1/cap1 mutant allowed assessment of impaired activation of cAMP signaling (Bahn and Sundstrom, supra), the effects of enhanced or hyperactivation of the cAMP-signaling pathway were not possible. Hypervirulence in the presence of hyperactivation of the cAMP-signaling pathway is found in C. neoformans where deletion of PKR1, which encodes the regulatory subunit of PKA, may lead to hypervirulence in animal models due to enhanced capsule production (D'Souza et al., supra). Additionally, detrimental effects of hyperactivation of the cAMP-signaling pathway were found for the plant pathogen, U. maydis. Enhanced activation of the cAMP-signaling pathway by constitutive activation of a Gα subunit or by mutation of the UBC1 gene in U. maydis led to normal tumor induction, however, fungal proliferation and development were reduced, indicating that detrimental effects may be at play (D'Souza and Heitman, supra; and Kruger et al., supra).
The C. albicans PDE1 gene was previously cloned and found to complement heat-shock sensitivity of S. cerevisiae pde2 mutants, but did not affect morphogenesis (Hoyer et al., 140 MICROBIOLOGY 1533-1542 (1994)). The present invention illustrates that disruption of PDE2 activates the cAMP-signaling pathway by limiting the ability to degrade cAMP in C. albicans, whereas overexpression down-regulates the cAMP-signaling pathway in C. albicans. The present invention is the first to show that a pde2/pde2 mutant may be hyperactive in forming germ tubes and production of HWP1, which may be accompanied by attenuated virulence. Further, the present invention is the first to describe genetic evidence showing that cAMP promotes true hyphae formation in C. albicans. The present invention also describes interference with CAP1 function, which has potential for providing novel strategies for interfering with candidiasis.
By defining the molecular events associated with the cAMP-signalling pathway through the identification of new genes that are associated with the cAMP-signalling pathway, the present invention has strong potential for identifying new and novel ways to interfere with candidiasis. The long-term medical benefits of the present invention may be the development of alternative or adjunctive therapies based on new knowledge about expression of PDE2 and CAP1 genes in C. albicans. 
Other objects, features and advantages of the present invention will become apparent from the following detailed description. The detailed description and the specific examples, however, indicate only preferred embodiments of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.